The present invention relates to synthetic peptides the sequences of which correspond to regions of immunologically important proteins of Human T-cell Lymphotropic Virus Type II (HTLV-2). These peptides are useful as diagnostic reagents for detecting the presence of antibodies to HTLV-2. The peptides may also be useful as immunogens in a vaccine composition to elicit antibodies against and prevent infection by HTLV-2.
HTLV-2 and Human T cell Lymphotropic Virus Type I (HTLV-1) are genetically and antigenically related members of a family of oncogenic retroviruses sharing a tropism for T lymphocytes and an association with lymphoproliferative diseases. Due to the degree of homology between HTLV-1 and HTLV-2, serological studies have been unable to differentiate between infection by HTLV-1 and HTLV-2. Unequivocal differentiation of HTLV-1 and HTLV-2 requires virus isolation and/or molecular identification.
HTLV-1 is endemic to southern Japan, and parts of the Caribbean, South America, Southeast United States and Central Africa. HTLV-1 is the etiologic agent of adult T-cell leukemia/lymphoma (ATL). Sarngadharan et al., Virology, (B.N. Fields et al., eds) pp. 1345-137,1 (1985). HTLV-1 is most prevalent in parts of Japan where approximately up to 15% of the population has been infected. Recently, HTLV-1 has been linked to the disease tropical spastic paraperesis (TSP) also known as HTLV-1 associated myelopathy (HAM) in Japan. Rodgers-Johnson et al., Lancet, 2:1247 (1985); Vernant et al., Ann. Neurol., 21:123 (1987). In the tropics, TSP is of the same magnitude and importance as the multiple sclerosis syndrome is in the western world. Marx, Science 236:1059-1061 (1987).
HTLV-2, in contrast, has not been definitely associated with human disease nor has it been shown to be endemic to any known population. The HTLV-2 virus has been isolated from two patients with T cell variants of hairy cell leukemia. Rosenblatt et al., New Engl. J. Med., 315:372-377 (1986). One of these HTLV-2 positive patients also had a co-existing CD8.sup.+ lymphoproliferative disorder. Rosenblatt et al., Blood, 71:363-369 (1988).
Significantly, intravenous drug abusers (IVDAs) previously thought to be infected with HTLV-1 have now been found to be infected by HTLV-2. It is now thought that HTLV-2 infection may be quite common among IVDAs. Tedder et al., Lancet, 2:15-128, (1984); and Robert-Guroff et al., J. Amer. Med. Assn., 255:3133-3137 (1986). This was recently supported by a study using PCR amplification which detected HTLV-2 related nucleotide sequences in a group of IVDAs from New Orleans. Lee et al., Science, 244:471-474 (1989). From serological studies reported from parts of the United States it has been shown that IVDAs may be infected with HTLV-1 and/or HTLV-2 with seropositivity rates as high as 24%. Gallo et al., J. Clin. Micro., 26:1487-1491 (1988).
Currently available tests for detection of viral infections rely on the detection of antibodies to the virus or portions thereof. Methods being developed for detecting HTLV infection, in general, will measure exposure to the virus by detecting and quantifying antibodies to HTLV antigens in blood, sera, and blood-derived products. Assays commonly used in diagnosis of other viral infections would be of immeasurable use in screening blood and blood products for previous exposure to HTLV-1 and/or HTLV-2. Due to the relatedness of the two viruses, it has been impossible to distinguish between infection by HTLV-1 or HTLV-2 using currently available immunological methods. Consequently, the virus is isolated from the patient and then distinguished on the basis of its nucleotide sequence. Often the virus must be propagated in vitro to provide enough material to test. Virus isolation is particularly difficult and dangerous in the case of IVDAs since they are often infected with human immunodeficiency virus (HIV) the causative agent of acquired immunodeficiency syndrome (AIDS).
Recently, the study of viral DNA has been aided by the use of polymerase chain reaction (PCR). However, PCR may still require virus isolation to obtain sufficient material.
An automated blood screening test format capable of readily detecting HTLV-2 infection and distinguishing HTLV-1 infection from HTLV-2 infection is critical to a supply of uninfected blood. Current methods of HTLV-1 screening cannot discern between HTLV-1 and HTLV-2 quickly and inexpensively.
The source of antigens for assays to detect HTLV-1 infection has until now included HTLV-1 proteins obtained from HTLV-1 infected T cell lines and antigens produced by recombinant DNA techniques. In theory, antigens for use in detecting HTLV-2 infection would be obtained from the same sources. The use of antigens obtained from these sources, however, has significant drawbacks in addition to their crossreactivity with HTLV-2.
The production of HTLV-2 per se in continuous cell lines must be performed in high risk (P3 containment) laboratories due to the danger to investigators who may become adversely exposed to the virus. In addition, protein antigens have been shown to give false negative and false positive results. For instance, enzyme linked immunosorbent assay (ELISA) tests utilizing whole virus HIV-1 antigens obtained from cell lines are prone to such errors. Gurtler et al., J. Virological Methods, 15:11-23 (1987). It is likely that similarly unreliable results will be obtained with cell-derived HTLV-2 antigens. Western blot analyses, for HTLV-2 detection using electroblotted whole virus antigens, may provide greater specificity but this method is laborious, time-consuming and not easily automated. Furthermore, since cells producing HTLV-2 are of human origin, viral antigens obtained from these cell lines, unless exhaustively purified, are likely to be contaminated with normal cellular antigens, such as HLA antigens, which could produce false positive reactions in an ELISA test.
Exhaustive purification of viral antigens from cell lines can also destroy immunogenicity of immunologically important proteins or otherwise inactivate antigens, thereby producing reagents that result in false negative reactions. In addition, false negative reactions using live virus derived antigens may occur because of steric hindrance whereby antibodies cannot react with their specific antigens because the reaction is blocked by the presence of other antigens and antibodies in the reaction mixture.
Proteins isolated from live virus can be unsuitable for vaccination due to the risk of contamination by whole virus or virus genomes.
ELISA tests to detect HTLV-2 infection may also employ immunologically important viral proteins produced by cloning portions of the HTLV-2 genome in various expression systems such as bacteria, yeast or vaccinia. The complete nucleotide sequence of HTLV-2 has been reported and the viral envelope glycoproteins and core proteins respectively encoded by the env and gag genes of HTLV-2, are apparently antigens recognized by antibodies in the sera of patients with HTLV-2 and HTLV-1 infections. Shimitohno et al., Proc. Natl. Acad. Sci. USA, 82:3101-3105 (1985).
Recombinant antigens purified from the host, may be used in diagnosis and as potential vaccine compositions as has been done for HIV-1 proteins. Cabradilla et al., Biotechnology, 4:128-133 (1986); Chang et al., Biotechnology, 3:905-909 (1985); Putney et al., Science, 234:1392-1395 (1986); and Kieny et al., Biotechnology, 4:790-795 (1986). As diagnostics, HTLV-2 antigens produced by recombinant DNA methods, however, will still have to be exhaustively purified to avoid false positive reactions in the ELISA due to any antibody reactivity to host antigens which are likely to contaminate the HTLV-2 antigen preparation unless exhaustively purified. Also, denaturation of HTLV-2 antigens during purification may destroy important antigenic regions.
In the case of vaccines, recombinant proteins purified from bacteria or yeast are often contaminated with bacterial or yeast proteins. Even minute amounts of these contaminants are capable of causing adverse reactions in patients.
Materials which approach 100% accuracy and specificity in diagnosis of HTLV-2 would be valuable given the nature of the diseases caused by HTLV-1 and possible diseases caused by HTLV-2 and the need for accurate type specific results.